Prime editing with genuine Cas9 nickases minimizes unwanted indels.

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing.

Targeted A-to-G base editing of chloroplast DNA in plants.

Chloroplast DNA (cpDNA) encodes up to 315 (typically, 120-130) genes, including those for essential components in photosystems I and II and the large subunit of RuBisCo, which catalyses CO fixation in plants. Targeted mutagenesis in cpDNA will be broadly useful for studying the functions of these genes in molecular detail and for developing crops and other plants with desired traits. Unfortunately, CRISPR-Cas9 and CRISPR-derived base editors, which enable targeted genetic modifications in nuclear DNA, are not suitable for organellar DNA editing, owing to the difficulty of delivering guide RNA into organelles.

Base editing in human cells with monomeric DddA-TALE fusion deaminases.

Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddA, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size.

Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.

Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g.

Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases.

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here, we present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNA-binding proteins, the split interbacterial toxin deaminase DddA, and a uracil glycosylase inhibitor (UGI), which catalyze targeted C-to-T base conversions without inducing unwanted small insertions and deletions (indels) in human cells. We assemble plasmids encoding ZFDs using publicly available zinc finger resources to achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA.

Pupil responses associated with the perception of gravitational vertical under directional optic flows.

This study assessed the pupil responses in the sensory integration of various directional optic flows during the perception of gravitational vertical. A total of 30 healthy participants were enrolled with normal responses to conventional subjective visual vertical (SVV) which was determined by measuring the difference (error angles) between the luminous line adjusted by the participants and the true vertical. SVV was performed under various types of rotational (5°/s, 10°/s, and 50°/s) and straight (5°/s and 10°/s) optic flows presented via a head-mounted display.

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities.

Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity.

Genome-wide target specificity of CRISPR RNA-guided adenine base editors.

Adenine base editors enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing, to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites containing inosines, the products of adenine deamination, we treat human genomic DNA with an adenine base editor 7.

Microsurgical Foraminotomy via Wiltse Paraspinal Approach for Foraminal or Extraforaminal Stenosis at L5-S1 Level : Risk Factor Analysis for Poor Outcome.

Objective: The purpose of this study was to present the outcome of the microsurgical foraminotomy via Wiltse paraspinal approach for foraminal or extraforaminal (FEF) stenosis at L5-S1 level. We investigated risk factors associated with poor outcome of microsurgical foraminotomy at L5-S1 level.

Methods: We analyzed 21 patients who underwent the microsurgical foraminotomy for FEF stenosis at L5-S1 level.

Postlaminectomy Bilateral Lumbar Intraspinal Synovial Cysts.

Lumbar intraspinal synovial cysts are included in the difference diagnosis of lumbar radiculopathy. Developing imaging modalities has result in increased reporting about these lesions. However, the case of bilateral new lumbar intraspinal synovial cysts after laminectomy has been rarely reported.